Ferredoxin Sequences Research Articles

Characterization of photosynthetic ferredoxin from the Antarctic alga Chlamydomonas sp. UWO241 reveals novel features of cold adaptation

The objective of this work was to characterize photosynthetic ferredoxin from the Antarctic green alga Chlamydomonas sp. UWO241, a key enzyme involved in distributing photosynthetic reducing power. We hypothesize that ferredoxin possesses characteristics typical of cold-adapted enzymes, namely increased structural flexibility and high activity at low temperatures, accompanied by low stability at moderate temperatures. To address this objective, we purified ferredoxin from UWO241 and characterized the temperature dependence of its enzymatic activity and protein conformation. The UWO241 ferredoxin protein, RNA, and DNA sequences were compared with homologous sequences from related organisms. We provide evidence for the duplication of the main ferredoxin gene in the UWO241 nuclear genome and the presence of two highly similar proteins. Ferredoxin from UWO241 has both high activity at low temperatures and high stability at moderate temperatures, representing a novel class of cold-adapted enzymes. Our study reveals novel insights into how photosynthesis functions in the cold. The presence of two distinct ferredoxin proteins in UWO241 could provide an adaptive advantage for survival at cold temperatures. The primary amino acid sequence of ferredoxin is highly conserved among photosynthetic species, and we suggest that subtle differences in sequence can lead to significant changes in activity at low temperatures.

CDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.)

Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

Identification of weak points prone for mutation in ferredoxin of <i> Trichomonas vaginalis</i>

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women. At present, metronidazole-resistant trichomoniasis is an infrequent but challenging problem with no universally successful treatment. Genetic mutation is believed to be an important factor leading to increasing drug resistance. Understanding the mutation status will help to design accurate strategies of therapy against mutant strains of T. vaginalis. The author performed a bioinformatic analysis to determine positions that tend to comply peptide motifs in the amino acid sequence of ferredoxin of T. vaginalis. Based on this study, the weak linkages in the studied protein can be identified and can be useful information for prediction of possible new mutations that can lead to drug resistance. In addition, the results from this study can be good information for further research on the diagnosis for mutants and new effective drug development.

Richard V. Eck (1922–2006): Bioinformatics: In the beginning

Richard V. Eck (1922–2006): Bioinformatics: In the beginning

Amino Acid Sequences of Ferredoxins from Atropa belladonna and Hyoscyamus niger: Their Similarities to Those in Other Tropane-Alkaloid-Containing Plants

The complete amino acid sequences of [2Fe-2S] ferredoxin from Atropa belladonna and Hyoscyamus niger have been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. These two ferredoxins exhibited 1-8 differences in their amino acid sequences compared to those of other tropane-alkaloid-containing plants (Scopolia japonica, Datura stramonium, D. metel, and D. arborea), and only 1 or 4 differences compared to S. japonica and D. arborea. In contrast, 9-23 differences were observed among the other solanaceous ferredoxins. This suggests that tropane-alkaloid-containing plants are closely related taxonomically.

Structure and dynamics of a predicted ferredoxin-like selenoprotein in Japanese encephalitis virus

Homologues of the selenoprotein glutathione peroxidase (GPx) have been previously identified in poxviruses and in RNA viruses including HIV-1 and hepatitis C virus (HCV). Sequence analysis of the NS4 region of Japanese encephalitis virus (JEV) suggests it may encode a structurally related but functionally distinct selenoprotein gene, more closely related to the iron-binding protein ferredoxin than to GPx, with three highly conserved UGA codons that align with essential Cys residues of ferredoxin. Comparison of the probe JEV sequence to an aligned family of ferredoxin sequences gave an overall 30.3% identity and 45.8% similarity, and was statistically significant at 4.9 S.D. ( P < 10 −6) above the average score computed for randomly shuffled sequences. A 3-dimensional model of the hypothetical JEV protein (JEV model) was constructed by homology modeling using SYBYL, based upon a high resolution X-ray structure of ferredoxin (PDB code: 1awd). The JEV model and the model from 1awd were subsequently subjected to molecular dynamics simulations in aqueous medium using AMBER 6. The solution structure of the JEV model indicates that it could fold into a tertiary structure globally similar to ferredoxin 1awd, with RMSD between the averaged structures of 1.8 Å for the aligned regions. The modeling and MD simulations data also indicate that this structure for the JEV protein is energetically favorable, and that it could be quite stable at room temperature. This protein might play a role in JEV infection and replication via TNF and other cellular stimuli mediated via redox mechanisms.

Establishment of a pure culture of the hitherto uncultured unicellular cyanobacterium Aphanothece sacrum, and phylogenetic position of the organism.

Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells approximately 6 microm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.

Eukaryotic genomes contain a [2Fe 2S] ferredoxin isoform with a conserved C-terminal sequence motif

Apicomplexan protists contain a single mitochondrial [2Fe2S] ferredoxin sequence (mtFd) with a highly conserved C-terminal motif, VDGxxpxPH, that distinguishes it from other mtFd, which have heterogeneous C-termini. This isoform of mtFd, called ‘type II ferredoxin’, is widespread in eukaryotes, some species having two isoforms and others possessing only one. Because of the known modulating role of the C-terminus of type I mtFd during association with itself and other interacting proteins, the presence of a conserved C-terminus in type II mtFd suggests it evolved either as a means for optimized homodimerization or to allow interaction with a highly conserved partner(s) that is yet to be defined.

Browsing gene banks for Fe2S2 ferredoxins and structural modeling of 88 plant-type sequences: an analysis of fold and function.

One-hundred-and-seventy-nine sequences of Fe2S2 ferredoxins and ferredoxin precursors were identified in and retrieved from currently available protein and cDNA databases. On the basis of their cluster-binding patterns, these sequences were divided into three groups: those containing the CX4CX2CXnC pattern (plant-type ferredoxins), those with the CX5CX2CXnC pattern (adrenodoxins), and those with a different pattern. These three groups contain, respectively, 139, 36, and 4 sequences. After excluding ferredoxin precursors in the first group, two subgroups were identified, again based on their cluster-binding patterns: 88 sequences had the CX4CX2CX29C pattern, and 29 had the CX4CX2CXmC (m not equal 29) pattern. The structures of the 88 ferredoxins with the CX4CX2CX29C pattern were modeled based on the available experimental structures of nine proteins within this same group. The modeling procedure was tested by building structural models for the ferredoxins with known structures. The models resulted, on average, in being within 1 A of the backbone root-mean-square deviation from the corresponding experimental structures. In addition, these structural models were shown to be of high quality by using assessment procedures based on energetic and stereochemical parameters. Thus, these models formed a reliable structural database for this group of ferredoxins, which is meaningful within the framework of current structural genomics efforts. From the analysis of the structural database generated it was observed that the secondary structural elements and the overall three-dimensional structures are maintained throughout the superfamily. In particular, the residues in the hydrophobic core of the protein were found to be either absolutely conserved or conservatively substituted. In addition, certain solvent-accessible charged groups, as well as hydrophobic groups, were found to be conserved to the same degree as the core residues. The patterns of conservation of exposed residues identified the regions of the protein that are critical for its function in electron transfer. An extensive analysis of protein-protein interactions is now possible. Some conserved interactions between residues have been identified and related to structural and/or functional features. All this information could not be obtained from the analyses of the primary sequences alone. Finally, the analysis of the sequences of the related subgroup featuring the CX4CX2CXmC (m not equal 29) cluster-binding pattern in the light of the structural and functional insights provided by the inspection of the mentioned structural database affords some hints on the functional features of ferredoxins belonging to this subgroup.

The Transit Sequence of Ferredoxin Contains Different Domains for Translocation across the Outer and Inner Membrane of the Chloroplast Envelope

Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross-linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence.

Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.

Amino acid sequence of ferredoxin from Physalis alkekengi var. francheti

The complete amino acid sequence of [2Fe-2S] ferredoxin from Physalis alkekengi var. francheti has been determined by automated Edman degradation of the entire Cm-protein and of the peptides obtained by trypsin and endoproteinase Asp-N digestions. This ferredoxin exhibited ten, ten, and nine differences respectively in the amino acid sequence, when compared with the ferredoxins of Datura stramonium, D. metel, and D. arborea, but 21–28 differences for other angiosperms, and 34–37 differences for fern and horsetails. These results are in harmony with the taxonomic position for these plants.

The petF region of the chloroplast genome from the diatom Thalassiosira weissflogii: sequence, organization and phylogeny

We report the cloning and characterization of a 1881 bp Thalassiosira weissflogii (Bacillariophyceae, Centrales) plastid genomic fragment containing the ferredoxin gene (petF) and several other genes. The organization of this fragment is exactly the same as its equivalent region from the plastid genome of another centric diatom, Odontella sinensis, and the sequences share high homology in both coding and non-coding regions. Traces of a probable vestigial protein-coding gene and of a partial trn gene are found just after the trnR gene in both diatoms as well as in the plastid genome of the red alga Porphyra purpurea, illustrating the close relationship between red algal and heterokont algal plastid genomes and arguing in favour of the origin of the latter by secondary endosymbiosis. In accordance with phylogenetic analyses of most plastid genes, phylogenetic trees inferred from 69 ferredoxin sequences show that both the green and the red algal plastid genes arose from cyanobacterial genes; our results furthermore suggest that each of these plastid lineages is related to different extant cyanobacterial genes, implying either that different endosymbiotic events were responsible for the origin of green and red plastids or that each lineage kept only one (and a different one) of several paralogous cyanobacterial genes present in the ancestral cyanobacterial genome.

The petF region of the chloroplast genome from the diatom Thalassiosira weissflogii: sequence, organization and phylogeny

We report the cloning and characterization of a 1881 bp Thalassiosira weissflogii (Bacillariophyceae, Centrales) plastid genomic fragment containing the ferredoxin gene (petF) and several other genes. The organization of this fragment is exactly the same as its equivalent region from the plastid genome of another centric diatom, Odontella sinensis, and the sequences share high homology in both coding and non-coding regions. Traces of a probable vestigial protein-coding gene and of a partial trn gene are found just after the trnR gene in both diatoms as well as in the plastid genome of the red alga Porphyra purpurea, illustrating the close relationship between red algal and heterokont algal plastid genomes and arguing in favour of the origin of the latter by secondary endosymbiosis. In accordance with phylogenetic analyses of most plastid genes, phylogenetic trees inferred from 69 ferredoxin sequences show that both the green and the red algal plastid genes arose from cyanobacterial genes; our results furthermore suggest that each of these plastid lineages is related to different extant cyanobacterial genes, implying either that different endosymbiotic events were responsible for the origin of green and red plastids or that each lineage kept only one (and a different one) of several paralogous cyanobacterial genes present in the ancestral cyanobacterial genome.

Cloning, characterization and transcriptional studies of ferredoxin genes from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and the thermophilic cyanobacterium Synechococcus vulcanus

Genes encoding ferredoxins from the transformable unicellular cyanobacterium Synechocystis sp. PCC 6803 and the thermophilic unicellular cyanobacterium Synechococcus vulcanus were cloned and sequenced. Southern blot hybridization detected a single copy of the ferredoxin sequences in both cyanobacterial genomes. Northern blot hybridization analyses indicated RNA transcripts of approximately 440 bases for Synechocystis sp. PCC 6803 and 460 bases for Synechococcus vulcanus. The accumulation of transcripts of the two cyanobacteria was three to four times greater in cells grown in iron‐rich medium than in cells grown in iron‐deficient medium.

Native-like beta-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues.

The conformational properties of protein fragments have been widely studied as models of the earliest initiation events in protein folding. While native-like alpha-helices and beta-turns have been identified, less is known about the factors that underly beta-sheet formation, in particular beta-hairpins, where considerably greater long-range order is required. The N-terminal 20 residue sequence of native ferredoxin I (from the blue-green alga Aphanothece sacrum) forms a beta-hairpin in the native structure and has been studied in isolation by NMR and CD spectroscopy. Local native-like interactions alone are unable to stabilize significantly a folded conformation of the 20-residue fragment in purely aqueous solution. However, we show that the addition of low levels of organic co-solvents promotes formation of native-like beta-hairpin structure. The results suggest an intrinsic propensity of the peptide to form a native-like beta-hairpin structure, and that the organic co-solvent acts in lieu of the stabilizing influence of tertiary interactions (probably hydrophobic contacts) which occur in the folding of the complete ferredoxin sequence. The structure of the isolated hairpin, including the native-like register of interstrand hydrogen bonding interactions, appears to be determined entirely by the amino acid sequence. The solvent conditions employed have enabled this intrinsic property to be established.

Purification and characterization of ferredoxin from Hydrogenobacter thermophilus strain TK-6.

Ferredoxin was purified from cells of Hydrogenobacter thermophilus strain TK-6. Purification was performed aerobically by the addition of octyl-beta-glucoside to the buffers. The purified ferredoxin had a molecular mass of 13,000 and contained a [4F3-4S] cluster. The protein had a long stretch at the N-terminal region; however, the sequence was not similar to the sequences of ferredoxins with a long stretch from Archaebacteria.

Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.)

Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5′ and 3′ regions

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 × 10 3 relative light units per second (RLU s −1) when assayed representing a stimulation of 18 000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5′ and 3′ ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.